Structural basis of DNA recognition and mechanism of quadruplex formation by the beta subunit of the Oxytricha telomere binding protein.
Interactions of the beta subunit of the Oxytricha nova telomere binding protein with the telomeric DNA sequences, d(T4G4)2 and dT6(T4G4)2, have been investigated in vitro using Raman and fluorescence spectroscopies. Raman difference spectra show that the beta subunit binds to both d(T4G4)2 and dT6(T4G4)2 but promotes the formation of a parallel-stranded quadruplex only in dT6(T4G4)2, thus demonstrating the importance of the telomeric 5' tail for in vitro recognition and guanine quadruplex formation. While d(T4G4)2 is not a suitable substrate for quadruplex promotion by the beta subunit, the Raman spectra reveal other structural rearrangements of this DNA strand upon beta subunit binding, including changes in guanine glycosyl torsion angles from syn to anti and disruption of carbonyl hydrogen-bonding interactions. The conformation of d(T4G4)2 in the beta:d(T4G4)2 complex is suggested as a plausible intermediate along the pathway to formation of the parallel-stranded guanine quadruplex. Fluorescence band shifts indicate that at least one of the two tryptophans of the beta subunit is shielded from solvent as a consequence of DNA binding in both the beta:dT6(T4G4)2 and beta:d(T4G4)2 complexes. However, the Raman spectra of these complexes suggest no significant changes in the beta subunit secondary structure attendant with DNA binding. A model for beta subunit binding by Oxytricha telomeric DNA sequences and a mechanism for quadruplex formation are proposed. A key feature of this model is the use of a telomeric hairpin secondary structure as the recognition motif.
Laporte
L
L
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City 64110, USA.
Thomas
G J
GJ
D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D006147:Guanine / Q000737:chemistry; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism*; D016813:Oxytricha / Q000737:chemistry* / Q000235:genetics; D011110:Polymorphism, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman; D013941:Thymine / Q000737:chemistry
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009841:Oligonucleotides; D006147:Guanine; D013941:Thymine